T-cell receptor binding to MHC-peptide complexes with different biological activities will be investigated to understand the relationship between TCR binding and activation, and thymocyte development. This 3roposal focuses on MHCp interaction with membrane-bound TCR. TCR-MHCp binding kinetics will be measured in relation to positive and negative selection, using tetramer binding studies. BIAcore analysis of the interaction between soluble TCR and MHCp will identify differences in the relative binding interactions when the TCR is isolated versus in the membrane in complex with CD3, and in the presence of co-receptors. The relationship between TCR binding and positive/negative selection will be investigated using natural positive selecting ligands and a series of ligands that span the border between 3ositive and negative selection. This will allow us to see how the TCR binding strength influences thymocyte selection. We will determine binding for MHCp ligands of different biological activity in the 3resence and absence of CD8. MHCp interaction kinetics with membrane-bound TCR and co-receptor will be studied using T cell membranes bound to BIAcore sensor chips, to measure the on and off rates for MHCp binding to TCR in the presence of the complete TCR:CD3 complex. The influence of CD8aa and ab on MHC class I:TCR binding and dissociation will be measured. Membranes from immature thymocytes will be used to understand how non-cognate CD8:MHCp interactions synergize with binding of TCR to MHCp, and the effect of sialylation on these interactions. Effects of altering lipid raft composition on TCR binding kinetics will be investigated. TCR dimerization and co-receptor interactions at the cell surface will be investigated using MHCp molecules and antibodies as cross-linking agents. Different strength ligands will be compared in order to determine how affinity and kinetics relates to the interaction between TCR and co-receptor. The potential dissociation of the TCR:CD3 complex after T-cell activation will be investigated using these techniques. TCR dimerization will be investigated in solution, using biophysical methods including FRET, analytical ultracentrifugation and BIAcore.